This proposal intends to identify and characterize the proteins found within the RNA interference (RNAi) pathway in C. elegans. RNAi is an important and highly conserved process that regulates developmental timing, RNA metabolism, and also serves as a host immune response to foreign RNA elements. Extensive genetic work has identified numerous genes involved in the RNAi pathway for this organism. However, only a small amount of biochemical data exists for these proteins, and many of the proteins directly involved in mRNA degradation remain unknown. This proposal intends to address this gap. Results from these studies will identify the proteins associated with mRNA target degradation and more clearly define their role within RNAi. This will be accomplished by isolating and characterizing the proteins that co-purify with RNAi activity from C. elegans cell extracts. In addition, proteins known to be involved in RNAi will be used to identify other proteins that interact with and influence in vitro RNAi activity. Finally, RNAi activity will be reconstituted from purified proteins in vitro.